Acid soluble proteins from micellar casein

ABSTRACT

The present invention relates to a milk fraction obtainable by acidification of micellar casein and separation from precipitated casein named acid soluble protein from micellar casein. It was found that the milk fraction and especially certain sub-fractions thereof are bioactive and promote GLP-1 release in vitro. Based on these results, acid soluble protein from micellar casein may be useful in the treatment and the prevention of diabetes type II, obesity and may further be added to formulas directed at other purposes addressing the gastro-intestinal tract.

BACKGROUND

The present invention relates to a method of obtaining acid solubleproteins of micellar casein, to fractions of the proteins, to theproteins for use as a medicament and to the use of the proteins in themanufacture of medicament or consumable products.

Diabetes mellitus is one of the most widespread diseases in the world.There are two major forms of diabetes mellitus: type I and type II. TypeI diabetic patients are characterised by an autoimmune destruction oftheir insulin secreting pancreatic beta cells. Type II diabeticpatients, which represent 90-95% of all diabetic patients, arecharacterized by development of insulin resistance in the peripheraltissues (principally liver and muscle), and an inappropriate insulinsecretion capacity.

People with type II diabetes are at high risk for serious long-termcomplications. These are essentially cardiovascular diseases, but alsoretinopathies, nephropathies and neuropathies.

Actual treatments for type II diabetes comprise several classes ofdrugs, which can be used alone or in combination with insulin, dependingon the amount of insulin still produced (sulfonylureas,thiazolidinediones, for example). Eventually, when no insulin isproduced any more, drug treatments may be replaced by injection ofinsulin alone.

Insulin biosynthesis and proinsulin gene expression are stimulated byGlucagon-Like Peptide-1 (GLP-1), expressed almost exclusively inendocrine intestinal cells. The important role of this secretagoguehormone is well summarised in “Glucagone-like peptide-1: a majorregulator of pancreatic b-cell function, R. Perfetti and P. Merkel,European Journal of Endocrinology (2000), 143, 717-725, which documentis incorporated herein by way of reference.

It was shown, for example, that after administration of intravenousGLP-1, the insulin secretory response type II diabetics was restored tothat of normal patients.

Furthermore, GLP-1 inhibits gastric motility, gastric acid secretion,gastric emptying and delays enzymatic breakdown and absorption ofnutrients. These affects are mostly preserved in both, type I and IIdiabetic patients

Moreover, GLP-1 was demonstrated to have an effect on satiety and islikely to be involved in decreased food intake.

GLP-1 is thus considered to be an ideal candidate for the treatment ofdiabetes.

Moreover, whenever one molecule of GLP-1 is liberated, one molecule ofGlucagon-Like Peptide-2 (GLP-2) is also liberated. Originating from onesingle mRNA, the mammalian proglucagon transcript. GLP-1 and GLP-2 arethus co-secreted in the gut.

GLP-2 inhibits gastric secretion and gastric motility. Chronic treatmentwith GLP-2 has beneficial trophic effects on the intestine, such asenhancing tissue mass and mucosal thickness, decreasing the rate ofenterocyte apoptosis, just to mention a few. An overview on GLP-1synthesis, secretion and biological activity may be derived from:Glucagon-Like Peptide 2, D. J. Drucker, The Journal of ClinicalEndocrinology and Metabolism, 2001, 86, 1759-64.

In WO 01/37850 (Société des Produits Nestlé) for the first time an invitro cell model to measure proglucagon gene expression and GLP-1secretion is described. The cell-line is called NCI-H716 and isdeposited, for example, under the ATCC number CCL-250. Accordingly,certain milk protein hydrolysates stimulate GLP-1 secretion.

WO 98/31239 describes a method for the selective hydrolysis of casein inthe presence of at least one further protein constituent. It ismentioned that the preparations so obtained are beneficial with respectto diabetes.

The objective of the present invention is one or several molecules thatstimulate the secretion of proglucagon derived hormones.

It is a further objective to find bio-active molecules that areconsidered nutritionally safe, for example, because they are naturallyoccurring in specific food resources.

Further objectives of the invention are to prevent or treat diabetestype II, to regulate glucose concentration in serum, to treat or preventbowel disorders characterized by injury and/or dysfunction of theintestinal mucosal epithelium, to increase the thickness and surfacearea of the intestinal mucosa, and/or to decrease appetite and foodintake.

It is also an objective of the present invention to improve GLP-1 and 2delivery in humans and mammals.

SUMMARY

Surprisingly, a protein fraction of milk, which is usually associatedwith or in close interaction to micellar casein, is capable ofstimulating the secretion of GLP-1. The very protein fraction may beliberated by exposing intact or enzyme treated casein to acidicconditions. Some of the proteins comprised in this fraction have neverbeen characterised so far.

Consequently, in a first aspect the present invention provides a methodof obtaining fractions of acid soluble proteins of micellar casein,comprising the steps of

separating micellar or enzyme-treated casein and whey proteins,

acidifying micellar casein or enzyme-treated casein to a pH below 6,

separating acid soluble proteins from casein, and

separating different fractions of acid soluble proteins.

In a second aspect the invention provides a sub-fraction of acid solubleproteins from micellar casein, characterised in that it is obtainable byhydrophobic interaction chromatography and that the fraction is elutedfrom a hydrophobic stationery phase by a mobile phase comprising 26.4 to36 vol.-% acetonitrile.

In a third aspect the invention provides a sub-fraction of acid solubleproteins from micellar casein, characterised in that the fraction isobtainable by hydrophobic interaction chromatography and that it iseluted from a hydrophobic stationery phase by a mobile phase comprising43.2 to 46.4 vol.-% acetonitrile.

In a fourth aspect, the present invention provides acid-soluble proteinsfrom micellar casein for use as a medicament or preventive ortherapeutic treatment of the human or animal body.

In a fifth aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments to enhance insulin secretion and/or proinsulingene expression.

In a sixth aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments for the prevention or treatment of diabetes typeI and/or II.

In a seventh aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments to increase GLP-1 and/or GLP-2 secretion and/orto regulate glucose concentration in blood.

In a further aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments for decreasing gastric emptying and acidsecretion.

In yet a further aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments for regulating appetite, decreasing food intakeand/or increasing satiety.

In another aspect, the present invention provides a consumable productcomprising any protein fraction or sub-fraction according to the presentinvention.

In another aspect, the present invention provides the use of acidsoluble proteins from micellar casein in the preparation of consumableproducts or medicaments for treating intestinal disorders characterisedby injury or dysfunction and/or to increase thickness and/or surfacearea of the intestinal mucosa.

An advantage of the present invention is that it provides naturallyoccurring active principles that are capable of stimulating secretion ofGLP-1.

Another advantage of the present invention is that the protein fractionin question may be easily isolated and supplied in sufficient amounts toany food product.

A further advantage of the present invention is that it providesnutritionally safe principles that may be useful in the treatment orprevention of diabetes type I and/or II, Crohn's disease, short bowelsyndrome, in the regulation of glucose levels in blood, and/or in theincrease of a satiety feeling and decrease in food intake.

Additional features and advantages are described herein, and will beapparent from, the following Detailed Description and the figures

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 compares the GLP-1 release in vitro stimulated by differentprotein fractions of bovine milk. The proteins were administered at 0.5mg/ml (shaded bars) and 5 mg/ml (dark bars), with the exception of theacid soluble proteins of micellar casein according to the invention,which showed the highest effect on GLP-1 release even at lowconcentration (shaded bar). Milk extracts were prepared in Krebs-RingerBalanced Buffer (KRBB), pH 7.4 comprising 0.2% BSA. The control consistsof the buffer (KRBB) and BSA (0.2%) only

FIG. 2 compares the GLP-1 release in vitro stimulated by differentsub-fractions of acid soluble proteins of micellar casein. Differentsub-fractions were administered in identical concentrations (30 μg/ml),and it is found that especially the sub-fractions 5, 7, 8, 10, and 11show a prominent effect on GLP-1 secretion. The total, which comprisesthe original mixture of all 14 fractions, was administered at muchhigher concentration (5 μg/ml).

FIG. 3 shows a HI-HPLC (hydrophobic interaction-high performance liquidchromatography) chromatogram of 14 fractions of acid soluble proteinsfrom micellar casein. The horizontal axis illustrates increasinghydrophobicity of acid soluble proteins from micellar casein, which werepartitioned into said 14 different sub-fractions. The vertical axisindicates the amount of protein eluted from the column, measured by UVat 214 nm.

DETAILED DESCRIPTION

Within the context of this specification the word “comprises” is takento mean “includes, among other things”. It is not intended to beconstrued as “consists only of”.

In the context of the present invention, the term consumable product isintended to encompass any nutritionally complete or supplementaryconsumable product. Hence, the composition may be consumed by humans,pets, such as cats and dogs, for example, and/or other animals. It may,be a bar, a snack, a nutritional formula, such as a liquid or powderedand reconstitutable formula, an infant or baby formula, an ice cream, adairy product, a confectionery product, or it may be a supplement or amedicament, which may, optionally, be added to another food product,such as the ones given above. It may also be a liquid product.

For example, if the food product is a nutritional formula, it may be anexclusive formula or a supplemental formula. An exclusive formula isusually consumed in an amount of 1.8 to 2.2 L daily by adults, and from0.6 to 1.4 L daily by infants.

If the formula is used supplementary, the daily amount is about ⅛ toabout 1/12 of the amounts for exclusive formulas, for example.

However, a consumable product according to the present invention is notrestricted to any product. It may be a food product itself or aningredient or component of any food product.

With respect to the method of acid soluble proteins of micellar casein,the separation of native or micellar casein from whey proteins, or ofenzyme treated casein from whey proteins, may be performed in many ways,such as by ultracentrifugation, or microfiltration, for example. Wheyproteins may also be separated from casein by selective removal ofspecific whey proteins, for example according to their hydrophobicproperties.

Acidification of micellar or enzyme treated casein is made by adjustingpH below 6, preferably below 5, more preferably below 4.8, for example,4.6. Any acid may be suitable, as long as it is food grade, such as HCl,acetic acid, and so forth.

Separation of acid soluble casein from micellar casein may again be doneby any suitable method, such as ultracentrifugation, filtration,decantation, and other.

Separation of different sub-fractions of acid soluble proteins may bedone according to hydrophobic properties, for example by hydrophobicinteraction chromatography (HIC), hydrophobic interaction liquidchromatography (HI-HPLC) and methods based on similar principles. Alsoother methods may be suitable, such as separation exploiting size orcharge properties of different fractions, for example.

With respect of the sub-fractions of acid-soluble proteins according tothe invention, they may be defined by their elution characteristics asgiven in Table 1. Table 1 shows that the fraction eluted in the range ofan acetonitrile concentration of 26.4 to 36 vol-%, corresponds to arange of 33 to 45 vol.-% of buffer B in a mixture consisting of Buffer Aand B as defined below Table 1.

The ranges of Buffer B and Buffer A concentrations above are sufficientto characterise the eluted protein fraction (Table 1). For the aboveelution ranges, preferably polystyrene-divinylbenzene beads are used asa stationary phase. Preferably, the product under the catalogue No. 15RPC TN 17-0727-02 from Amersham is used as a stationery phase.

In particular, the pH of elution according to the above characteristicsmay generally be in the range of 1.8 to 2.2, preferably about 2. This pHis mainly defined by the amount of trifluoroacetic acid TFA).

In addition to presumably unknown substances, it is likely thatsub-fraction 6, 7 and 8 comprise PP8 (proteose peptone), fractions 8 and9 comprise PP8 and PP5 (proteose peptones), fraction 10 compriseslactoferrin and fraction 11 comprises β-lactoglobulin.

Thus, in preferred embodiments according to the invention, thesub-fractions according to the present invention are sub-fractions 5, 6,7, 8, 10, and 11 as characterised in Table 1 by elution at specificbuffer or acetonitril concentration ranges. Sub-fraction 5,specifically, may be re-tested additionally, because some cytotoxicproperties are suspected.

A possible way of working the present invention is by first isolatingthe specific milk protein fractions reported herein. This may be done inany adequate way and there are at least a few procedures suitable.

For example, acid-soluble proteins from micellar casein may be obtainedby isolating casein from milk or skimmed milk (or derivatives) byultracentrifugation. Accordingly, skimmed milk is ultra-centrifuged at30,000 to 90,000 g or up to 110,000 g for 45 to 90 min, for example. Thesediments may be recovered, constituting micellar casein (or:non-soluble proteins).

The micellar casein may be washed by dispersing it in CaCl₂ 2mM/NaCl0.9% and ultra-centriuged as above.

Acid-soluble proteins from casein may then be obtained by acidificationof the washed micellar casein, as described above. For example, micellarcasein (non-soluble protein) may be dispersed in sodium acetate buffer20 mM pH 4.6. Due to casein buffering behaviour, a pH of about 4.6 maybe achieved by acetic acid (or other suitable acid) addition.

The solution may then be centrifuged (9,000 to 15,000 g, 20 to 50 min,for example) and the supernatant may be collected as acid-solubleprotein from micellar casein.

Another way of obtaining acid-soluble proteins from micellar casein maybe derived from: Protein composition of micellar casein obtained bycross-flow micro-filtration of skimmed milk, R. Jost, R. Brandsma, S.Rizvi, International Dairy Journal 9 (1999) 389-90.

Accordingly, native casein is obtained by micro-filtration instead ofultra-centrifugation. Hence, micro-filtration membranes with pore sizesin the range of 0.1-0.2 μm are used to separate the micellar phase ofmilk from its serum phase by a purely physical process.

For example, skimmed milk warmed to 50° C. may be filtered through a1P19-40 Tetra-Laval 1.4 μm Membralox®D module to reduce its bacterialload. Then it may be warmed to 55° C. and separated on a Tech-Sep 1S 151micro-filtration unit equipped with Carbosep® M14 (0.14 μm pore size)membranes. The milk may thus be fractionated into a retentate and apermeate stream, operating in a concentration mode. After reachingconcentration factor 3 (cf3), diafiltration may be initiated by additionof demineralized water. The retentate obtained after diafiltration of 6initial volumes of water (cf3/df6) constitutes micellar casein.

The acid-soluble parts of micellar casein may then be obtained byacidification of rediluted retentate with hydrochloric (or other, suchas acetic) acid to pH 4.6, followed by separation of curd bycentrifugation. As a result, 3-5% of the total N may be separated fromthe curd as acid-soluble proteins of micellar casein.

In so doing a protein fraction comprising, amongst other, lactoferrin,serum albumin, immunoglobulin H- and L-chains, and a protein having anelectrophoretic mobility similar to β-lactoglobulin may be obtained.

Furthermore, the fraction may comprise proteose peptone component 5(β-casein 1-105/1-107) and proteose peptone component 8 fast.

Other components of the acid-soluble proteins of micellar casein aredefined by the present invention.

Of course, the above methods just serve as examples for the isolation orpurification of acid-soluble proteins from micellar casein. The skilledperson may easily envisage other methods for obtaining them.

For example, it is possible that acid-soluble proteins of micellarcasein remain attached at least partially to casein after enzymetreatment (for example, rennet). In this case, acid-soluble protein ofmicellar casein may be obtainable by treating milk enzymatically,followed by recovering the curd formed by the coagulated casein. Theacid-soluble fraction of micellar casein may then be at least partiallyisolated from the curd by acidification as above, for example.

Such a procedure would replace the isolation of micellar casein bycentrifugation or ultrafiltration, in so far as the acid-solubleproteins of micellar casein mainly remain associated to the cleavedcasein fractions.

It may also be possible to recover the acid-soluble proteins of micellarcasein by acidifying milk or skimmed milk. Then the proteins accordingto the present invention must be separated from the acid whey, thelatter further comprising other soluble milk components comprised inacid whey (mainly α-lactablumin, β-lactoglobulin, lactoferrin). This maybe done by any suitable separation technique suitable to removeselectively the whey fraction mentioned above, for example by HIC or ionexchange chromatography.

The present invention is based on the surprising cognition that acidsoluble proteins of micellar casein comprise bio-active principles thatare suitable to promote GLP-1 and (therewith connected) GLP-2 release.

However, further sub-fractionation of the acid soluble proteins frommicellar casein and screening reveals that some sub-fractions of theacid soluble proteins of micellar casein are preferred.

Further separation and isolation of more effective sub-fractions may beachieved by hydrophobic interaction chromatography (HIC), or byhydrophobic interaction high performance liquid chromatography(HI-HPLC), reversed-phase high-performance liquid chromatography(RP-HPLC), and the like, all of which are based on the same separationprinciples, for example.

The principles of HIC are known to the skilled person. Generally,samples are loaded onto an equilibrated column (stationery phase)comprising a hydrophobic material retaining the samples. The hydrophobicmaterial may be, for example, macroporous crosslinked polystyrene,commercialised as Amberlite Xad 16 (XAD 16 from Rohm and Hass), forexample. 15 RPC TN 17-0727-02 (polystyrene-divinylbenzene) from Amershamor equivalents may also be used.

Before the protein fraction according to the invention is loaded ontothe column, the latter may be equilibrated with a buffer. After thefraction is loaded, a buffer or a mixture of buffers (mobile phase) maybe run over the column, whereby the mixture of buffers varies and mayhave, therefore, varying properties of eluting protein sub-fractionsaccording to their hydrophobicity from the column.

Separation of whey proteins according to this method is described in:“Simultaneous separation and quantitation of the major bovine wheyproteins including proteose peptone and caseinomacropeptide byreversed-phase high-performance liquid chromatography onpolystyrene-divinylbenzene”, D. F. Elgar et al., Journal ofChromatography A 878 (2000) 183-196.

The protein sub-fractions eluted from the column may accurately bedescribed by the composition of the buffer mixture or acetonitrilecontent that effected their elution from the stationery phase.

For example, acid soluble proteins from micellar casein may be loadedonto a column filled with polystyrene-divinylbenzene beads (15 RPC TN17-0727-02 from Amersham), a buffer A may be defined as 0.1 vol.-%trifluoroacetic acid (TFA) in water and a buffer B may be defined as 80vol-% acetonitril and 0.85 vol.-% TFA in water.

Then, the mixing and transport of buffers A and B may be controlled by aspecific system, for example a FPLC (Fast Protein Liquid Chromatography)UNICORN station (Pharmacia, Amersham), and flown through the column.

The eluted protein sub-fraction may be defined by an elution range ofmixing-ratios of the above mentioned buffers A and B. Using the specificbuffer composition, the elution moment or interval of a proteinsub-fraction may be described simply by the relative amount ofacetonitrile present at the moment of elution of a protein fractionaccording to the invention. It should be noted, however, that theelution order is pH dependent.

Table 1 below defines 14 acid soluble protein sub-fractions of theacid-soluble protein from micellar casein according to the invention bya vol.- percentage range of buffer B, or a range in acetonitrile, withinwhich sub-fractions according to preferred embodiments of the presentinvention are eluted. TABLE 1 Sub-fractions of acid soluble proteinsfrom micellar casein defined by hydrophobic interaction chromatography.% B Buffer % B Buffer % acetonitrile % acetonitrile fraction start endstart end 1 20 22.5 16 18 2 22.5 26 18 20.8 3 26 30 20.8 24 4 30 33 2426.4 5 33 36 26.4 28.8 6 36 39.5 28.8 31.6 7 39.5 43.5 31.6 34.8 8 43.545 34.8 36 9 45 49 36 39.2 10 49 54 39.2 43.2 11 54 58 43.2 46.4 12 5867.5 46.4 54 13 67.5 81.5 54 65.2 14 81.5 100 65.2 80Buffer A: Water TFA 0.1%Buffer B: Acetonitrile/Water/TFA (80%/19.15%/0.85%; v/v)Column: Source 15 RPC Amersham (matrix: Polystyrene/divinyl benzene),column volume (CV = 100 ml)Gradient: starting from 20% B Buffer, sample was injected after 1 columnvolume (CV), then gradient increased up to 75% B in 15 CV then 2 CV toreach 100% B Buffer

If desirable, the sub-fractions may be concentrated by evaporation,ultrafiltration, or dialysed to eliminate organic solvent before drying,for example by vacuum-, freeze-, spray-, fluidised bed-, oven-, or anyother suitable drying process.

Sub-fractions 5, 6, 7, 8, 9, 10, and 11 are specifically effective inpromoting GLP-1 release in vitro and constitute a preferred embodimentin the sense of the present invention. Sub-fractions 5, 7, 8 and 11constitute an even more preferred embodiment.

Sub-fraction 5 has been shown to have a toxic effect on cells of an invitro model, hence this fraction may prove to have diminishedapplicability in humans or animals.

Acid soluble proteins of micellar casein comprise biologically activeprinciples that enhance GLP-1 secretion in an in vitro model.

Therefore, the fraction or selected sub-fractions may be used forregulation of any process dependent or controlled by GLP-1, GLP-2 orinsulin. Examples are the prevention or treatment of diabetes I or II,regulation of blood glucose concentration, inhibition of gastricmotility and secretion, decrease gastric emptying rate of liquids andsolids, decrease small intestine transit, inhibition of smooth muscleactivity, decrease meal-induced glucose excursions, delay of enzymaticbreakdown and absorption of nutrients in the intestines, decrease ofappetite, decrease of food intake, and the like.

For example, the fraction may be used to decrease overall digestive andabsorptive activity of a human or an animal.

The protein fraction may be added to consumable products or medicaments.Examples of consumable products are nutritional formulas, infantformulas or clinical formulas. Other examples are drinks, for exampleshelf stable, chilled or ready-to-drink beverages. The fractions may beadded to other food products, such as chocolate, bars, cereals, dairyproducts, ice cream, frozen food, pet food, coffee, capsule, tablet, forexample.

The following examples are given by way of illustration only and in noway should be construed as limiting the subject matter of the presentapplication. Percentages and parts are by weight unless otherwiseindicated.

EXAMPLE 1 Isolation of Acid Soluble Protein from Micellar Casein

Bovine milk was obtained from a local market (Switzerland, Toni lait,2000-02-01).

Cream was extracted from whole milk by centrifugation between 1,000 and4,500 g. The selectivity of this step was improved by increasingacceleration up to 13,600 g using fixed-angle rotor Sorvall GS3 (9,000rpm during 30 minutes). Starting from 2,200 ml of whole milk, 90 g ofcream were recovered in the top layer.

Then, 250 μl of CaCl₂ 200 mM were added to 250 ml of skimmed milk toreach a final 2 mM concentration. This milk was ultra-centrifuged (6tubes containing 42.1 g of skimmed milk) 1 hour in a fixed-angle rotor45 TI (Beckman L8-60M ultracentrifuge; 32,000 rpm corresponding to100,000 g in the middle of the tube) to separate whey from non-solublemicellar casein.

Micellar casein (24 g) was dispersed in 220 ml CaCl₂ 2 mM/NaCl 0.9% andultra-centrifuged as above. The 22 g washed micellar casein recoveredwere dispersed in CaCl₂ 2 mM/NaCl 0.9% and volume adjusted to 250 ml. Itwas aliquoted, labeled non-soluble proteins (micellar casein) andfrozen.

Starting from 190 g whole milk the deposit of washed non-solubleproteins (17 g) was dispersed (Potter) in 40 ml sodium acetate buffer 20mM pH 4.6.

Due to casein buffering behavior, pH (6.5) was adjusted to 4.6 by aceticacid addition, and volume was adjusted to half initial milk volume (90ml) by acetate buffer addition. Solution was then centrifuged (12,000 g,30 min) and supernatant (77 g) was labeled Acid-soluble protein frommicellar casein.

For the purpose of Example 3 below, the sample was frozen by immersionin liquid nitrogen and stocked at −20° C.

EXAMPLE 2 GLP-1 Release Promoted by different Milk Fractions

Material and Methods

MH (meat hydrolysate) and EAH (egg albumin hydrolysate) were purchasedfrom Sigma. Matrigel was from BD Bioscience. CGMP was obtained asdescribed in WO 9853702.

Fractionation steps to obtain skimmed milk, sweet whey, acid whey, acidcasein, soluble proteins, non-soluble proteins (micellar casein) wereadapted from conventional milk processes (see: Alais C. 1984 Science dulait, Principe des Techniques Laitières, 4ème édition, SEPAIC, Paris,29-35, 159-178). Centrifugation was performed at higher accelerationrates and non-soluble fractions were washed to increase selectivity andseparation efficacy.

Acid casein is casein collected from sediments after acid treatment ofskimmed milk.

Soluble proteins are proteins recovered in aqueous solution afterultracentrifugation of skimmed milk for 1 h at 100,000 g (see alsoExample 1), non-soluble protein (micellar casein) being the partrecovered in sediments thereafter.

Acid-soluble proteins from micellar casein were taken from Example 1,basically obtained by acidifying and centrifuging the resulting acidcasein of the above paragraph.

NCI-H716 human intestinal cell line (ATCC number: CCL-251) was culturedat 37° C. in a humidified incubator containing 5% CO₂. Forproliferation, cells were grown in suspension in RPMI 1640 medium (LifeTechnologies Inc) supplemented with 10% FBS, 100 IU/ml penicillin, 100μg/ml streptomycin and 2 mM L-glutamine. For secretion studies, cellswere plated on Matrigel-coated plates, and incubated 2 days in DMEM (lowglucose) (Life Technologies Inc) supplemented with 10% FBS, 100 IU/mlpenicillin, 100 μg/ml streptomycin and 2 mM L-glutamine.

Two days before the experiment, cells were plated at 1 millioncells/well in 12-wells plates. On the day of the experiment, cells werewashed once with HBSS (Hank's Balanced Salt Solution; Life TechnologiesInc), and incubated during two hours at 37° C. in the presence of thedifferent protein solutions. Test proteins were dissolved in 1 ml ofKRBB (Krebs-Ringer Balanced Buffer) pH 7.4 containing 0.2% BSA (fractionV; Sigma). At the end of the incubation period, supernatant wasrecovered in 10 μl PMSF 200 mM and immediately frozen at −80° C.

Protein solutions were added in two concentrations, 0.5 and 5 mg proteinper ml medium.

Results

FIG. 1 shows the effect of different milk protein fractions at differentconcentrations (0.5 and 5 mg/ml) on GLP-1 release in vitro. Acid-solubleproteins from micellar casein obtained according to Example 1 have thehighest impact on GLP-1 release even at low concentration (0.5 mg/ml).

In conclusion, acid-soluble proteins from micellar casein, obtainable bythe method according to Example 1 comprise bio-active principles. Thesemay be used in the prevention of disease or therapy of the human oranimal body. The release of GLP-1 suggests usefulness and industrialapplicability of these principles in the prevention and treatment ofdiabetes and obesity, for example.

EXAMPLE 3 Sub-fractionation of Acid Soluble Protein from Micellar Casein

A hydrophobic interaction liquid chromatographiy (HIC) was performed asfollows. A HR16×50 column filled with 100 ml Source 15 RPC TN 17-0727-02(polystyrene-divinylbenzene) was connected to a FPLC system controlledby a UNICORN station (Amersham Pharmacia Biotech). 15 ml of HClacid-soluble fraction (see Example 1) were thawed 20 min in a water-bathat 37° C., mixed by vortexing and centrifuged 1 min at 13,000 rpm in a5415 Eppendorf centrifuge. After filtering on a 0.45 μm Millipore filter(306/GSWP04700.GS), 10 ml of this preparation was injected.

Chromatographic conditions were: A buffer: TFA 0.1% in water (2,000 mlof miliQ water filtered on a 0.45 μm Millipore system, plus 2 ml TFA(Sigma 91699, 100 ml)); B buffer: acetonitrile 80%, TFA 0.85% (400 ml ofmiliQ water filtered on a 0.45 μm Millipore system, plus 1,600 mlacetonitrile, degassed in an ultrasound bath during 15 minutes, andfinally additionned with 1.7 ml TFA).

Column was equilibrated with 20% B buffer. Then, after one column volume(CV), sample was injected, B buffer increased to 75% in 15 CV and to100% in 2.5 CV. At the end, gradient decreased to 20% B buffer in 0.4CV. Flow rate was fixed at 3 ml/min.

96 fractions of 18 ml were collected in plastic tubes. Fractions werekept at −20° C. After HPLC analysis, the 96 collected tubes were pooledin 14 fractions by similitude of HPLC profile and concentrated byevaporation before lyophilisation for subsequent screening.

UV absorption at 215 nm was recorded, the corresponding HIC profile isgiven in FIG. 3

The 14 sub-fractions of acid-soluble proteins from micellar casein arecharacterised by way of their moment of elution from the column and thecorresponding Acetonitrile concentration (hydrophobicity) in Table 1 ofthe description.

EXAMPLE 4 GLP-1 Release Promoted by Sub-fractions of Example 3

The sub-fractions obtained in Example 3 were screened for GLP-1 releasepromoting capacity according to the experimental design as set out inExample 2. All sub-fractions were tested at 30 μg/ml media, except for“total”, comprising all sub-fractions, which was tested at 500 μg/ml.

It is mentioned that in this experiment, hydrophobic chromatographypools were tested at a concentration 16-17 times lower than the initialacid-soluble proteins from micellar casein of Example 2 (30 μg/mlcompared to 5001 μg/ml).

The result is shown in FIG. 2. Nearly all fractions caused an increaseof GLP-1 release in vitro. Extraordinarily strong increase is found withsub-fractions 5, 7, 8, but also with 9, 10, and 11 at very low proteinconcentrations (30 μg protein/ml media).

It was found, however, that sub-fraction 5 has toxic effect on thecell-line.

In conclusion, sub-fractions 7, 8, 9, 10, and 11 comprise bio-activemolecules or principles. These may serve as medicaments, especially inthe treatment of diabetes type II, possibly also type I or obesity. Thevarious other effects of GLP-1 in the human or animal body explainfurther applications of the sub-fractions as given in the claims.

EXAMPLE 5 A nutritional Formula Comprising Acid-Soluble Proteins fromMicellar Casein

A nutritional formula comprising, in percent by weight of dry matter,14% protein, 62% carbohydrate, 18% fat and 3.2% minerals and vitaminsaccording to recommended values (315 kJ/dl), is completed with acidsoluble protein from micellar casein according to Example 1.

The acid soluble protein is added in physiologically effective amounts.In a complete, exclusive formula (intended consumption is 2 1 per day)the final concentration was adjusted to 0.1-0.5 mg/ml of the formula. Informula useful as a supplement to other nutrition (intended consumption:2 dl per day) the concentration was adjusted to 1-5 mg/ml formula.Higher doses are used according to circumstance and individualrequirements.

EXAMPLE 6 A nutritional Formula Comprising Sub-fractions Acid-SolubleProteins from Micellar Casein

The formula of Example 5 is enriched with protein of sub-fractions 7 and8 obtained in Example 3 instead of protein according to Example 1.

The amount of dry matter of the sub-fractions in the nutritional formulais adjusted to be 5-25 μg/ml liquid formula for a complete formula (seeExample 5), and 50-250 μg/ml for a daily dose of 2 dl (supplement).

A high dose formula comprising the sub-fractions was also prepared, (10times concentrated with respect to the 2 dl supplement above). Theconcentration of the proteins according to the invention was 0.5-2.5mg/ml in 2 dl.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

1. A method of obtaining fractions of acid soluble proteins of micellarcasein, comprising the steps of separating micellar or enzyme-treatedcasein and whey proteins, acidifying micellar casein or enzyme-treatedcasein to a pH below 6, separating acid soluble proteins from casein,and separating different sub-fractions of acid soluble proteins.
 2. Asub-fraction of acid soluble proteins from micellar casein, obtained byhydrophobic interaction chromatography, the fraction being eluted from ahydrophobic stationery phase by a mobile phase comprising 26.4 to 36vol.-% acetonitrile.
 3. A sub-fraction of acid soluble proteins frommicellar casein, the fraction obtained by hydrophobic interactionchromatography and being eluted from a hydrophobic stationery phase by amobile phase comprising 43.2 to 46.4 vol.-% acetonitrile.
 4. Amedicament for the treatment of a human or animal body comprising anacid-soluble protein from micellar casein.
 5. The acid soluble proteinsaccording to claim 2, wherein the acid soluble proteins compriseproteins selected from the group consisting of proteose peptones 5,proteose peptones 8, β-lactoglobulin, lactoferrin, serum albumin,immunoglobulin and mixtures thereof.
 6. A method for enhancing insulinsecretion and/or proinsulin gene expression comprising the steps ofadministering to an individual a composition including an acid solubleprotein from micellar casein.
 7. A method for treating diabetescomprising the steps of administering to an individual having diabetes acomposition including acid soluble proteins from micellar casein.
 8. Amethod for increasing GLP-1 and/or GLP-2 secretion and/or to regulateglucose concentration in blood of an individual comprising the steps ofadministering to an individual a composition including acid solubleproteins from micellar casein.
 9. A method for decreasing gastricemptying and acid secretion of an individual comprising the steps ofadministering to an individual a composition including acid solubleproteins from micellar casein.
 10. A method for regulating appetite,decreasing food intake and/or increasing satiety of an individualcomprising the steps of administering to an individual a compositionincluding acid soluble proteins from micellar casein.
 11. A method fortreating intestinal disorders characterised by injury or dysfunctionand/or to increase thickness and/or surface area of the intestinalmucosa of an individual comprising the steps of administering to anindividual a composition including acid soluble proteins from micellarcasein.
 12. (canceled)
 13. The method of claim 7 wherein the diabetes istype I.
 14. The method of claim 7 wherein the diabetes is type II. 15.The acid soluble proteins according to claim 3 wherein the acid solubleproteins comprise proteins selected from the group consisting ofproteose peptones 5, proteose peptones 8, β-lactoglobulin, lactoferrin,serum albumin, immunoglobulin and mixtures thereof.
 16. The acid solubleproteins according to claim 4 wherein the acid soluble proteins compriseproteins selected from the group consisting of proteose peptones 5,proteose peptones 8, β-lactoglobulin, lactoferrin, serum albumin,immunoglobulin and mixtures thereof.
 17. A method for preventingdiabetes comprising the steps of administering to an individual at riskof same a composition including acid soluble proteins from micellarcasein.